EP3555620A1 - Procédé de détermination de la présence d'un antigène cible dans un échantillon de tumeur - Google Patents

Procédé de détermination de la présence d'un antigène cible dans un échantillon de tumeur

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Publication number
EP3555620A1
EP3555620A1 EP17825147.6A EP17825147A EP3555620A1 EP 3555620 A1 EP3555620 A1 EP 3555620A1 EP 17825147 A EP17825147 A EP 17825147A EP 3555620 A1 EP3555620 A1 EP 3555620A1
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EP
European Patent Office
Prior art keywords
antigen
antibody
tumor
seq
antigen binding
Prior art date
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Pending
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EP17825147.6A
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German (de)
English (en)
Inventor
Martina GEIGER
Christian Klein
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of EP3555620A1 publication Critical patent/EP3555620A1/fr
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to a new cell-based assay for determining antigen expression in primary tumor samples.
  • the method further relates to the determination of antigen and protease expression in primary tumor samples.
  • the method allows robust determination of antigen and/or protease expression without the need to digest the tumor samples.
  • the method further allows for selection of antibodies and for selection of protease-cleavable linkers for the treatment of tumors.
  • antibody based therapies have evolved over the last 15 years and represent now a valuable combination or alternative to chemotherapeutic approaches in the treatment of tumors.
  • antibody therapies target specific antigens on cancer cells thus allowing a more side directed treatment thereby reducing the side effects on healthy tissue.
  • various assays are required to identify the best candidates to bring into clinical trials and eventually to the market.
  • the antibodies In a first early preclinical phase, the antibodies have to be generated and analyzed for their target- specificity, as well as their affinity to the target and functionality.
  • Binding properties can be analyzed using various protein-protein interaction assays, such as FRET-based methods, Surface Plasmon Resonance (SPR), fluorescence-activated cell sorting (FACS) or Alpha ScreenTM. Functionality is generally tested in various cell-based assays designed to mimic the physiological situation as close as possible to identify the best candidates to be tested in animal models before entering clinical trials. These functional assays are commonly carried out using primary cells, tumor cell lines or reporter cells that are designed to express a reporter upon activation of a specific pathway. However, cell lines or primary cells in culture may not be a comprehensive model of tumor tissue which usually is a more complex three dimensional assembly of cells.
  • Tumor samples are much more difficult to assess directly because of several obstacles.
  • Tumors contain, besides the tumor cells, also a complex extracellular matrix environment, partially or completely generated by excretion of extracellular matrix components by cells in the tumor.
  • Extracellular matrix may restrain penetration of antibodies into the tumor tissue but may also interfere with accessability of cell surface targets on the tumor cells.
  • extracellular components may also contain additional antigenic or functional elements which may be valuable for targeted immunotherapy.
  • Bispecific constructs comprising a binding moiety capable of recogniting the tumor cells and an effector moiety activating immune cells have shown promising results. However, in some instances it might be necessary to conceil the effector moiety until delivery of the bispecific molecules to the tumor to reduce unspecific systemic side effects.
  • One approach to accomplish this is to conceal the effector moiety with an anti-idiotypic binding moiety capable of reversible binding the effector moiety and connecting the concaling moiety to the bispecific immunotherapeutic antibody using protease cleavable linkers.
  • Tumors especially malignant tumors, are well known to comprise proteases not expressed or not present in their active form in healthy adult tissue. Constructs with protease-cleavable linkers can therefore be used to target the activity of novel classes of bispecific antibodies to the tumor tissue. With increasing complexity of constructs for immunotherapy, the requirements to assays for measuring binding and functionality of antibodies in a comprehensive setup increase as well. Binding assays on cells in culture may not be sufficient to model the complex environment in tumor tissue.
  • the inventors of the present invention developed a novel assay which combines the assessment of binding and functionality of antibodies and antibody like constructs directly on tumor samples, such as e.g., tumor biopsies.
  • This novel assay is useful for example for screening or characterization purposes in early development of new antibody constructs as well as for selecting suitable antibodies for treatment of cancer.
  • This new assay represents a valuable tool for screening binding and targeted functionality in tumor samples which will allow identifying the best constructs at an early stage in the development of the drug candidate and to identify suitable treatments for patients.
  • an in vitro method for determining the presence of a target antigen in a tumor sample comprising the steps of:
  • reporter cells comprising a reporter gene under the control of a
  • a bispecific antibody comprising:
  • the target antigen is expressed by the tumor cells.
  • the expression of the reporter gene is indicative for binding of the first antigen binding moiety to the target antigen.
  • the bispecific antibody of step iii) additionally comprises c) a masking moiety covalently attached to the second antigen binding moiety through a protease-cleavable linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety.
  • a protease cleaves the protease-cleavable linker, wherein the second antigen binding moiety is unconcealed.
  • the protease is expressed by the tumor cells.
  • the expression of the reporter gene is indicative for protease expression in the tumor sample.
  • the tumor sample is a tumor tissue sample, in particular a biopsy from a patient.
  • the tumor sample is not digested. In one embodiment, the tumor sample is digested, in particular by in particular by collagenase or hyaluronidase.
  • the tumor sample contains dead cells, in particular more than 10% of dead cells.
  • protease expression is indicative for a malignant tumor.
  • the signal transducing cell surface receptor is functionally linked to a response element.
  • the response element controls the expression of the reporter gene.
  • the response element is part of the NF- ⁇ pathway.
  • the response element comprises at least one DNA repeat with a DNA sequence of SEQ ID NO: 68, 69, 70, 71 or 72.
  • the response element comprises a DNA sequence of SEQ ID NO 73, 74, 75 or 76.
  • the reporter gene is coding for a fluorescent or a luminescent protein.
  • the reporter gene is coding for green fluorescent protein (GFP) or luciferase.
  • the reporter cells comprise the DNA sequence coding for the reporter gene under the control of the response element, and the DNA sequence coding for signal transducing cell surface receptor.
  • the reporter cells comprise at least one DNA repeat with a DNA sequence of SEQ ID NO: 68, 69, 70, 71 or 72, wherein the DNA repeat is operatively linked to the reporter gene and wherein the reporter gene is expressed upon binding of the second antigen binding moiety to the signal transducing cell surface receptor.
  • the second antigen binding moiety is capable of specific binding to CD3.
  • the protease-cleavable linker comprises a protease recognition sequence.
  • the protease recognition sequence is selected from the group consisting of:
  • VHMPLGFLGPGRSRGSFP SEQ ID NO:46
  • VHMPLGFLGPRQARVVNG (SEQ ID NO:50);
  • VHMPLGFLGPPMAKK (SEQ ID NO:57);
  • the protease is selected from the group consisting of metalloproteinase, serine protease, cysteine protease, aspartic proteases, and cathepsin protease.
  • the metalloproteinase is a matrix metalloproteinase (MMP), particularly MMP9 or MMP2.
  • MMP matrix metalloproteinase
  • the serine protease is Matriptase.
  • the masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety. In one embodiment, the masking moiety is covalently attached to the light chain variable region of the second antigen binding moiety.
  • the masking moiety is an anti-idiotypic scFv.
  • the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker.
  • the first and the second antigen binding moieties are conventional Fab molecules comprising a common light chain.
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, optionally via a peptide linker.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker.
  • the bispecific antibody comprises a third antigen binding moiety capable of specific binding a tumor antigen.
  • the third antigen binding moiety is a conventional Fab molecule, or a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
  • the third antigen binding moiety is identical to the first antigen binding moiety.
  • the bispecific antibody additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • the Fc domain is an IgG, specifically an IgGl or IgG4, Fc domain.
  • the Fc domain is a human Fc domain.
  • the target antigen is a cell surface receptor. In one embodiment, the target antigen is FolRl.
  • the target antigen is a peptide bound to a molecule of the human major histocompatibility complex (MHC).
  • MHC human major histocompatibility complex
  • the peptide has an overall length of between 8 and 100, preferably between 8 and 30, and more preferred between 8 and 16 amino acids.
  • the binding to the target antigen and the expression of the protease are determined in the same vial.
  • an in vitro method for selecting a bispecific antibody for the treatment of a tumor wherein the bispecific antibody comprises:
  • a second antigen binding moiety capable of specific binding to a signal transducing cell surface receptor
  • the method comprises determining the presence of a target antigen in a tumor sample according to the method as described herein and wherein the bispecific antibody is selected for treatment of the tumor if expression of the reporter gene is detected.
  • FIG. 1A Jurkat-NFAT reporter cell line (Promega) is a human acute lymphatic leukemia reporter cell line with a NFAT promoter, expressing human CD3s. If the CD3 binder of the TCB binds the tumor target and the CD3 (cross- linkage is necessary) binds CD3s the Luciferase expression can be measured in Luminescence after addition of One-Glo substrate (Promega).
  • FIG. IB Masking the CD3 binder with a protease-cleavable linker only induces Jurkat-NFAT reporter gene activation if the protease expressed by the tumor can cleave the linker.
  • FIG. 2A ID 8364. 16D5 TCB, classic format, anti ID CH2527 scFv 4.32.63 MMP9-MK062 Matriptase site N-terminally fused to CD3.
  • FIG. 2B ID 8363. 16D5 TCB, classic format, anti ID CH2527 scFv 4.32.63 Cathepsin S/B site N-terminally fused to CD3.
  • FIG. 2C ID 8409. anti ID CH2527 scFv 4.32.63 non cleavable linker CD3 16D5 Fc.
  • FIG. 2D ID 6298.
  • FIG. 2E ID 6182 and 7235.
  • DP47GS TCB sf CHO W(9a).
  • FIG 2F ID 8408.
  • Figure 3 depicts CE-SDS analysis of different bispecific CD3 binders.
  • FIG. 3B CE-SDS analysis of the TCB 8363 depicted in FIG.
  • Figure 4 depicts the Jurkat NFAT activation assay according to FIG. 1 using HeLa (4A) and Skov-3 (4B) cells as target cells. Each point represents the mean value of triplicates. Standard deviation is indicated by error bars.
  • the FolRl TCB black triangles pointing down
  • rhMatriptase/ST14 pretreated protease-activated TCB 8364, grey filled squares
  • the masked TCB containing a GS non cleavable linker, grey triangles pointing up
  • the non-targeted TCB control empty triangle pointing down
  • the dotted line shows the Luminescence of target cells and effector cells without any TCB.
  • FIG. 5 depicts the Jurkat-NFAT activation assay with benign primary tumor sample and FolRl TCBs.
  • Jurkat NFAT reporter cells are activated after co-incubation with FolRl TCB (6298) in 24 well plate with cell culture inserts.
  • Protease-activated FolRl TCBs 8363, 8364, 8408) and control TCBs (8409, 7235) do not induce Luciferase expression.
  • the dotted line indicates the baseline Luminescence for Jurkat NFAT cells co-incubated with tumor.
  • Figure 6 depicts the Jurkat-NFAT activation assay with malignant primary tumor samples and FolRl TCBs.
  • Jurkat NFAT reporter cells are activated after co-incubation with FolRl TCB (6298) and protease-activated FolRl TCB containing MMP9-Matriptase cleavage site (8364) in 96 well plate with Matrigel. Protease-activated FolRl TCBs (8363, 8408) and control TCBs (8409, 7235) do not induce Luciferase expression. The dotted line indicates the baseline Luminescence for Jurkat NFAT cells co-incubated with tumor.
  • FIG. 7 depicts the Jurkat-NFAT activation assay with patient-derived xenografts.
  • FIG. 7A deptics a schematic of the p95Her2-TCB (SEQ ID NOs: 77. 78, 79, 80).
  • FIG. 7B depics quantitative analysis of p95Her2 expression of patient-derived xenografts (PDX) or human breast cancer samples.
  • FIG. 7C depict immunohistological staining of PDXs for HER2 or p95HER2.
  • FIG. 7D depics a correlation analysis of the quantitative IHC -based analysis and the Jurkat-NFAT activation with corresponding samples.
  • acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody or ligand) and its binding partner (e.g., an antigen or a receptor).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
  • amino acid mutation as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide.
  • Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids.
  • Particular amino acid mutations are amino acid substitutions.
  • non-conservative amino acid substitutions i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred.
  • Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3- methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G 329 , P329G, or Pro329Gly.
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • antibody herein is used in the broadest sense to a molecule that specifically binds an antigenic determinant and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • an antigen binding domain refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • an antigen binding moiety refers to a polypeptide molecule that specifically binds to an antigenic determinant.
  • an antigen binding moiety is able to direct the entity to which it is attached (e.g., a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant.
  • an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen.
  • Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region.
  • the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art.
  • Useful heavy chain constant regions include any of the five isotypes: ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ .
  • Useful light chain constant regions include any of the two isotypes: ⁇ and ⁇ .
  • an “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antibody which provides interaction with the antigen.
  • the antigen binding site of an antibody comprises amino acid residues from the "complementarity determining regions" (CDRs).
  • CDRs complementarity determining regions
  • a native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
  • the term "antigen-binding site of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the antigen-binding portion of an antibody comprises amino acid residues from the CDRs.
  • "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined.
  • the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties.
  • CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop".
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • scFv fragments see e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993).
  • Single- domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g., U.S. Patent No. 6,248,516 Bl).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage).
  • antigenic determinant is synonymous with “antigen” and “epitope”, and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.
  • Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the proteins referred to as antigens herein can be any native form of the proteins from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the antigen is a human protein.
  • the term encompasses the "full-length", unprocessed protein as well as any form of the protein that results from processing in the cell.
  • the term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants.
  • Exemplary human proteins useful as antigens include, but are not limited to: FolRl and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 60 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1 for the cynomolgus [Macaca fascicularis] sequence).
  • the bispecific molecule of the invention binds to an epitope of CD3 or a target cell antigen that is conserved among the CD3 or target antigen from different species.
  • the bispecific molecule of the invention binds to CD3 and FolRl.
  • bispecific antibody denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168).
  • Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol, 148(5): 1547- 1553 (1992)); using "diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci.
  • a bispecific antibody is able to specifically bind to at least two distinct antigenic determinants.
  • a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
  • the expressions "cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transfectants” and “transfected cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a rabbit variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies”.
  • Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244.
  • crossover Fab molecule also termed “Crossfab” is meant a Fab molecule wherein either the variable regions or the constant regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region and the heavy chain constant region, and a peptide chain composed of the heavy chain variable region and the light chain constant region.
  • the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain” of the crossover Fab molecule.
  • the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.
  • a "conventional" Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant regions (VH-CH1), and a light chain composed of the light chain variable and constant regions (VL-CL).
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion immune complex-mediated antigen uptake by antigen presenting cells
  • down regulation of cell surface receptors e.g. B cell receptor
  • B cell activation e.g. B cell activation
  • the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof.
  • Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
  • the terms engineer, engineered, engineering, particularly with the prefix “glyco-”, as well as the term “glycosylation engineering” are considered to include any manipulation of the glycosylation pattern of a naturally occurring or recombinant polypeptide or fragment thereof.
  • Glycosylation engineering includes metabolic engineering of the glycosylation machinery of a cell, including genetic manipulations of the oligosaccharide synthesis pathways to achieve altered glycosylation of glycoproteins expressed in cells. Furthermore, glycosylation engineering includes the effects of mutations and cell environment on glycosylation. In one embodiment, the glycosylation engineering is an alteration in glycosyltransferase activity. In a particular embodiment, the engineering results in altered glucosaminyltransferase activity and/or fucosyltransferase activity.
  • epitope includes any polypeptide determinant capable of specific binding to an antibody.
  • epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain.
  • Fc domain or "Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl- terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • a "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
  • a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
  • first and second with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the bispecific antibody unless explicitly so stated.
  • a “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.
  • FR Framework or “FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • VH or VL
  • full length antibody “intact antibody”
  • whole antibody are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • the term "functionality of an antibody or ligand” refers to the biological activity of an antibody or ligand, e.g. the ability of an antibody or ligand to elicit a cellular response. For example through binding to a target antigen, the antibody activates or suppresses a cell signaling pathway, i.e. activates of inhibits the function of the target antigen. For example, the antibody to be tested binds to a receptor activating the NF-KB pathway and through this binding a response element in the cell nucleus is activated. When linking this response element to a reporter gene, the activation can be easily monitored in the assay of the invention.
  • the term "functionality" also includes the effector functions of an antibody, e.g.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation; as well as activation of T cells.
  • B cell receptor cell surface receptors
  • fused is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
  • High-throughput screening shall be understood to mean that a relatively large number of different antibody or ligand candidates can be analyzed for binding and functionality with the novel assay described therein. Typical such high-throughput screening is performed in multi-well microtiter plates, e.g. in a 96 well plate or a 384 well plate or a plates with 1536 or 3456 wells.
  • the terms "host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • the term "human antibody” as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.)
  • a "human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup is subgroup III as in Kabat et al., supra.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • Other forms of "humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops").
  • native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3).
  • Exemplary CDRs CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3.
  • Hypervariable regions are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions.
  • CDRs complementarity determining regions
  • Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody.
  • Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody.
  • One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself.
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.
  • CDRs generally comprise the amino acid residues that form the hypervariable loops.
  • CDRs also comprise "specificity determining residues,” or "SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.
  • Exemplary a-CDRs (a-CDR- Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3.
  • HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
  • idiotype-specific polypeptide refers to a polypeptide that recognizes the idiotype of an antigen-binding moiety, e.g., an antigen-binding moiety specific for CD3.
  • the idiotype-specific polypeptide is capable of specifically binding to the variable region of the antigen-binding moiety and thereby reducing or preventing specific binding of the antigen-binding moiety to its cognate antigen.
  • the idiotype-specific polypeptide can function as a masking moiety of the molecule.
  • anti-idiotype antibodies or anti-idiotype-binding antibody fragments specific for the idiotype of anti- CD3 binding molecules are disclosed herein.
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region.
  • each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region.
  • VL variable region
  • CL constant light
  • the heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG), or ⁇ (IgM), some of which may be further divided into subtypes, e.g., ⁇ (IgGl), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), al (IgAl) and a2 (IgA2).
  • the light chain of an immunoglobulin may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
  • An "individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • ligand refers to any molecule that is able to bind to another molecule.
  • Example of ligand molecules include, but are not limited to peptides, proteins, carbohydrates, lipids, or nucleic acids.
  • Preferred ligands to be analysed with the assay described herein are peptides or proteins that are capable of binding to a target antigen. Usually such target antigen is a cell surface receptor.
  • a "modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer.
  • a modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits.
  • a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively.
  • (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same.
  • the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution.
  • the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • naked antibody refers to an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel.
  • the naked antibody may be present in a pharmaceutical formulation.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3).
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • NF- ⁇ refers to the "nuclear factor kappa-light-chain-enhancer of activated B cells” and is a transcription factor which is implicated in the regulation of many genes that code for mediators of apoptosis, viral replication, tumorigenesis, various autoimmune diseases and inflammatory responses.
  • NFKB is present in almost all eukaryotic cells. Generally, it is located in the cytosol in an inactive state, since it forms a complex with inhibitory kappa B ( ⁇ ) proteins.
  • ⁇ inhibitory kappa B
  • IKK IKB kinase
  • IKK is an enzyme complex which consists of two kinases and a regulatory subunit. This complex phosphorylates the IKB proteins, which leads to ubiquitination and therefore degradation of those proteins by the proteasome. Finally, the free NFKB is in an active state, translocates to the nucleus and binds to the ⁇ DNA elements and induces transcription of target genes.
  • NF- ⁇ pathway refers to the stimuli that lead to modulation of activity of NF-KB. For example activation of the Toll-like receptor signaling, TNF receptor signaling, T cell receptor and B cell receptor signaling through either binding of a ligand or an antibody result in activation of NF- ⁇ .
  • a "receptor of the NF- ⁇ pathway” refers to a receptor which can trigger the modulation of activity of NF- ⁇ : Examples of a “receptor of the NF- ⁇ pathway” are Toll-like receptors, TNF receptors, T cell receptor and B cell receptor.
  • Non-limiting examples of antibodies that upon binding to its target result in modulation of the activity of NF-KB are anti-CD3 antibodies, anti-CD40 antibodies, anti-DR5 antibodies, anti-DR4 antibodies, anti-41BB antibodies, anti-Ox40 antibodies and anti-GITR antibodies.
  • Examples of ligands that upon binding to its target result in modulation of the activity of NF- ⁇ are OX40 ligand, 4- IBB ligand or CD40 ligand.
  • No substantial cross -reactivity means that a molecule (e.g., an antibody) does not recognize or specifically bind an antigen different from the actual target antigen of the molecule (e.g. an antigen closely related to the target antigen), particularly when compared to that target antigen.
  • an antibody may bind less than about 10% to less than about 5% to an antigen different from the actual target antigen, or may bind said antigen different from the actual target antigen at an amount consisting of less than about 10%, 9%, 8% 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1% antigen different from the actual target antigen.
  • the antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” (see, US 2008/0069820, for example).
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • proteolytic enzyme refers to any proteolytic enzyme that cleaves the linker at a recognition site and that is expressed by a target cell, e.g. by a tumor cell. Such proteases might be secreted by the target cell or remain associated with the target cell, e.g., on the target cell surface.
  • proteases include but are not limited to metalloproteinases, e.g., matrix metalloproteinase 1-28 and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, e.g., urokinase-type plasminogen activator and Matriptase, cysteine protease, aspartic proteases, and members of the cathepsin family.
  • metalloproteinases e.g., matrix metalloproteinase 1-28 and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33
  • serine proteases e.g., urokinase-type plasminogen activator and Matriptase
  • cysteine protease aspartic proteases
  • members of the cathepsin family members of the cathepsin family.
  • Protease-activatable refers to a T cell activating bispecific molecule having reduced or abrogated ability to activate T cells due to a masking moiety that reduces or abrogates the T cell activating bispecific molecule's ability to bind to CD3.
  • proteolytic cleavage e.g., by proteolytic cleavage of a linker connecting the masking moiety to the T cell activating bispecific molecule
  • binding to CD3 is restored and the T cell activating bispecific molecule is thereby activated.
  • protein with intrinsic fluorescence refers to a protein capable of forming a highly fluorescent, intrinsic chromophore either through the cyclization and oxidation of internal amino acids within the protein or via the enzymatic addition of a fluorescent co- factor.
  • protein with intrinsic fluorescence includes wild-type fluorescent proteins and mutants that exhibit altered spectral or physical properties. The term does not include proteins that exhibit weak fluorescence by virtue only of the fluorescence contribution of non-modified tyrosine, tryptophan, histidine and phenylalanine groups within the protein. Proteins with intrinsic fluorescence are known in the art, e.g. green fluorescent protein (GFP),), red fluorescent protein (RFP), Blue fluorescent protein (BFP, Heim et al.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • BFP Heim et al.
  • CFP Heim et al. 1996; Tsien 1998
  • YFP yellow fluorescent variant
  • YFP Ormo et al. 1996; Wachter et al. 1998
  • Sapphire Tin-excitable green fluorescent variant
  • cyan-excitable green fluorescing variant known as enhanced green fluorescent protein or EGFP (Yang et al. 1996) and can be measured e.g. by live cell imaging (e.g. Incucyte) or fluorescent spectrophotometry.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
  • recombinant human antibodies have variable and constant regions in a rearranged form.
  • the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • a "reporter gene” means a gene whose expression can be assayed.
  • a “reporter gene” is a gene that encodes a protein the production and detection of which is used as a surrogate to detect indirectly the activity of the antibody or ligand to be tested.
  • the reporter protein is that protein encoded by the reporter gene.
  • the reporter gene encodes an enzyme whose catalytic activity can be detected by a simple assay method or a protein with a property such as intrinsic fluorescence or luminescence so that expression of the reporter gene can be detected in a simple and rapid assay requiring minimal sample preparation.
  • enzymes whose catalytic activity can be detected are Luciferase, beta Galactosidase, Alkaline Phosphatase. Luciferase is a monomeric enzyme with a molecular weight (MW) of 61 kDa.
  • luciferin acts as a catalysator and is able to convert D-luciferin in the presence of Adenosine triphosphate (ATP) and Mg2+ to luciferyl adenylate.
  • ATP Adenosine triphosphate
  • Mg2+ Mg2+
  • pyrophosphate (PPi) and adenosine monophosphate (AMP) are generated as byproducts.
  • the intermediate luciferyl adenylate is then oxidized to oxyluciferin, carbon dioxide (C0 2 ) and light.
  • Oxyluciferin is a bioluminescent product which can be quantitatively measured in a luminometer by the light released from the reaction.
  • Luciferase reporter assays are commercially available and known in the art, e.g.
  • Luciferase 1000 Assay System and ONE-GloTM Luciferase Assay System refers to the binding of a masking moiety or idiotype- specific polypeptide to an antigen-binding moiety or molecule such as to prevent the antigen-binding moiety or molecule from its antigen, e.g., CD3.
  • This concealing is reversible in that the idiotype- specific polypeptide can be released from the antigen- binding moiety or molecule, e.g., by protease cleavage, and thereby freeing the antigen- binding moiety or molecule to bind to its antigen.
  • single-chain refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.
  • one of the antigen binding moieties is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
  • the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.
  • T cell activation refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • a cytotoxic T lymphocyte selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • Some of the bispecific antibodies molecules as described herein are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art and described herein.
  • target cell antigen refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma.
  • target antigen refers to any cell surface antigen that can be targeted by an antibody or fragment thereof. It also refers to the receptor that can be targeted by a ligand.
  • a “response element” refers to a specific transcription factor binding element, or cis acting element which can be activated or silenced on binding of a certain transcription factor.
  • the response element is a cis-acting enhancer element located upstream of a minimal promotor (e.g. a TATA box promotor) which drives expression of the reporter gene upon transcription factor binding.
  • a “signal transducing cell surface receptor” as used herein is a cell surface receptor localized on the surface of reporter cells as described herein capable of transducing an extracellular signal, e.g. binding of an antigen binding moiety to the signal transducing cell surface receptor, to an intracellular signaling cascade resulting with expression of a reporter gene.
  • signal transducing cell surface receptors are Toll-like receptors, TNF receptors, T cell receptor and B cell receptor or recombinant versions or fragments thereof.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g. "tetravalent” or "hexavalent”).
  • variable region or variable domain refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self -replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • Antibodies used in the the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).
  • Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of full length antibodies to which further antigen-binding sites (e.g., single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) are linked via one or more pep tide-linkers.
  • the antibodies can be full length from a single species, or be chimerized or humanized.
  • the inventor developed a robust assay suitable for high-throughput format which enables determination of antigen expression in tumors and/or functional activity of bispecific antibodies in tumor cells, particularly in primary tumor samples.
  • Functionality of the antibody e.g. the the biological activity of an antibody such as the ability of an antibody to elicit a cellular response
  • reporter cell lines which have a reporter gene expressed upon activation of a response element.
  • said reporter gene is selected from a gene encoding for a fluorescent protein (e.g. green fluorescent protein, GFP) and/or a gene encoding for an enzyme whose catalytic activity can be detected (e.g. Luciferase).
  • an in vitro method for determining the presence of a target antigen in a tumor sample comprising the steps of:
  • reporter cells comprising a reporter gene under the control of a
  • a bispecific antibody comprising:
  • the target antigen can be an antigen expressed by the tumor cells, and is usally located on the cell surface of the tumor cells.
  • the target antigen is expressed by the tumor cells.
  • the tumor cells naturally express the target antigen.
  • the target antigen is located on the surface of the tumor cells.
  • the target antigen is a cell surface receptor. Accordingly, the bispecific antibody binds to the target antigen on the cell surface of the tumor cells.
  • the target antigen is selected from the group consisting of CEA, Her2, TYRP, EGFR, MCSP, STEAP1, WT1 and FolRl.
  • the target antigen is FolRl.
  • the target antigen is not limited to proteins located on the cell surface but may also derive from polypeptides or proteins which are termporarly or permanently located intracellularly. In such cases, the target antigen deriving from an intracellular polypeptide or protein is presented on the cell surface, in particular on the cell surface of the tumor cells.
  • the target antigen is a peptide bound to a molecule of the major histocompatibility complex (MHC). In one embodiment, the MHC is human MHC. In one embodiment, the peptide bound to a molecule of the MHC has an overall length of between 8 and 100, preferably between 8 and 30, and more preferred between 8 and 16 amino acids.
  • the target antigen derives from a protein which is exclusively or mainly expressed in tumor tissue.
  • the protein is an intracellular protein and the peptide is generated by the MHC-I or MHC-II pathway and presented by a MHC class I or MHC class II complex.
  • the peptide is generated by the MHC-I pathway and presented by a MHC class I complex.
  • the tumor cells are mammalian cells, preferably human or primate cells.
  • the tumor cells derive from a tumor sample, in particular from a biopsy from a patient.
  • the tumor sample is a biopsy from a human patient.
  • the tumor cells bear the target antigenic determinant.
  • the tumor cells derive from a patient suffering from a proliferative disorder, particularly cancer.
  • Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • the tumor cells derive from a biopsy of a human patient.
  • the tumor sample is a biopsy of a human patient. The tumor sample can be assessed without dissociation of the tumor sample or tumor tissue into single cells.
  • the tumor sample is not digested prior to determining the presence of a target antigen according to the method of the present invention.
  • the tumor sample is cut, in particular using a razor blade.
  • the tumor sample is digested prior to determining the presence of a target antigen according to the method of the present invention.
  • the tumor sample is digested, in particular by collagenase or hyaluronidase.
  • the bispecific antibody binds to the signal transducing cell surface receptor on the reporter cells wherein the response element activates the expression of the reporter gene. Accordingly, the reporter gene in the reporter cells is expressed upon binding of the first antigen binding moiety to the target antigen and binding of the signal transducing cell surface receptor to the reporter cells. In one embodiment, the expression of the reporter gene is indicative for binding of the first antigen binding moiety to the target antigen. Surprisingly, no expression or only very low expression of the reporter gene occurs without binding of the first antigen binding moiety to the target antigen on the tumor cells.
  • the binding of the bispecific antibody to the target antigen can be determined qualitatively, i.e.
  • the absence of reporter gene expression is defined by a certain threshold, i.e. after deduction of any background signal.
  • the background signal is usually determined by performing the assay with all reagents but the antibody to be tested or in absence of the tumor cells.
  • the binding of the antibody or ligand to the target antigen can be determined quantitavely, i.e. the level or strength of binding can be determined with the method according to the invention. Towards this end the antibody is tested in different concentrations and the half maximal effective concentration (EC50) is determined.
  • EC50 refers to the concentration of the antibody or ligand at which the antibody binds halfway between the baseline and maximum after a specified exposure time.
  • the EC50 of the dose response curve therefore represents the concentration of the antibody where 50% of its maximal binding is observed.
  • the KD dissociation constant
  • the bispecific antibody binds to the signal transducing cell surface receptor.
  • the binding of the antibody to the signal transducing cell surface receptor elicits a cellular response which results in a modulation of the activity of the response element, either directly or through a cascade of cell signalling.
  • the response element is a DNA element which can be silenced or activated by transcription factors or the like.
  • Response elements are known in the art and are commercially available, e.g. in reporter vectors.
  • the response element comprises DNA repeat elements and is a cis-acting enhancer element located upstream of a minimal promotor which drives expression of a reporter gene upon transcription factor binding. Examples for response elements and their transcription factors useful herein are mentioned in the below table:
  • AP1 Monitoring induction of the activator protein 1(AP) and the stress-activated protein kinase/J un N-terminal kinase (SAPK/JNK) signal transduction pathway.
  • SAPK/JNK stress-activated protein kinase/J un N-terminal kinase
  • API (2) Monitoring the induction of the protein kinase C (PKC) signal transduction pathway, as well as related pathways such as the MAPK pathway.
  • PKC protein kinase C
  • the androgen receptor functions as a steroid-hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from, accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes.
  • CRE(l) Measuring transcriptional activity of cAMP binding protein (CREB).
  • CRE cAMP response element
  • JNK Jun N-terminal kinase
  • PKA protein kinase A
  • E2F1 E2F2, E2F3, E2F4, E2F5.
  • the E2F protein family plays a crucial role in the control of cell cycle and action of tumor suppressor proteins and is also a target of the transforming proteins of small DNA tumor viruses. These proteins bind preferentially to retinoblastoma protein pRB and mediate both cell proliferation and p53-dependent independent apoptosis.
  • ELK1 Measuring transcriptional activity of ELK 1.
  • ELK1 is a member of the Ets family of transcription factors and of the ternary complex factor (TCF) subfamily. Proteins of the TCF subfamily form a ternary complex by binding to the serum response factor and the serum reponse element in the promoter of the c-fos proto-oncogene.
  • ELK l is a nuclear target for the ras- raf-MAPK signaling cascade.
  • Cytokines bind and induce receptor dimerization at the cell surface, causing the receptor itself to be
  • the phosphorylated receptor then acts as a docking site for gamma
  • STATl is phosphorylated, dimerizes and translocates to the nucleus activation
  • GATA GATA Measuring transcriptional activity of globin transcription factor (GATA) family.
  • GATA globin transcription factor
  • the GATA family of transcription factors contains six zinc-finger binding proteins that regulate differentiation and cell proliferation. GATA family members are involved in hematopoietic, cardiac and gut development.
  • GRE glucocorticoid response element
  • HIF-1 Measuring transcriptional activity of hypoxia inducible factor- 1 (HIF-1).
  • HIF-1 binds to the hypoxia-response element and activates genes involved in angiogenesis, glucose metabolis, cell proliferation/survival and invasion/metastasis.
  • HSE heat shock factor
  • IRF-1 Measuring transcriptional activity of interferon regulatory factor 1.
  • IRFl is a member of the interferon regulatory transcription factor (IRF) family. IRFl serves as an activator of interferons alpha and beta transcription, and in mouse it has been shown to be required for double- stranded RNA induction of these genes.
  • Jak/STAT-mediated signal transduction pathways Signaling molecules, including type I (IFN-a and -b) and type II (IFN-g) interferons, induce signaling by binding receptors and causing receptor dimerization at the cell surface. This dimerization causes the receptor itself to be phosphorylated and act as a docking site for transcription factors, including STAT1 and STAT2. The STAT proteins are then phosphorylated, dimerize and translocate to the nucleus, where the STAT1 and STAT2 heterodimer regulates transcription by binding to the IFN-stimulated response element (ISRE).
  • ISRE IFN-stimulated response element
  • MEF-1 Measuring transcriptional activity of myogenic factor 3 (MYODl ).
  • NFAT nuclear factor of activated T-cells
  • NFKB Monitoring the activation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFKB) signal transduction pathway.
  • NFKB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines, oxidant-free radicals, ultraviolet irradiation, and bacterial or viral products.
  • Activated NFKB translocates into the nucleus and stimulates the expression of genes involved in a wide variety of biological functions.
  • p53 Monitoring p53-mediated signal transduction pathways.
  • p53 is a tumor suppressor that plays a crucial role in a number of cellular processes, including the suppression of cell proliferation after DNA damage.
  • RAR Monitoring the induction of the retinoic acid response element (RARE).
  • RXR retinoid X receptors
  • RXR-mediated signal transduction pathway Retinoid X receptors (RXRs) and retinoic acid receptors (RARs) are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation. These receptors exert their action by binding, as homodimers or
  • heterodimers to specific sequences in the promoters of target genes and regulating their transcription.
  • Spl Measuring transcriptional activity of Spl .
  • Spl is a sequence-specific
  • S l was initially identified as a HeLa cell derived factor that selectively activates in vitro transcription from the SV40 promoter and binds to the multiple GC boxes in the 21 -bp repeated elements in SV40.
  • Spl has been described as a ubiquitous transcription factor that is required for the constitutive and inducible expression of a variety of genes, such as in cell cycle or mammalian development.
  • SRE serum response element
  • MAP mitogen-activated protein
  • Statl p84/p91 Measuring transcriptional activity of signal transducer and activator of transcription 1.
  • Statl is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein can be activated by various ligands including interferon-alpha, interferon-gamma, EGF, PDGF and I...- .
  • Stat4 Measuring transcriptional activity of signal transducer and activator of transcription 4.
  • Stat4 protein encoded by this gene is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell, nucleus where they act as transcription activators. This protein is essential for mediating responses to IL-12 in lymphocytes, and regulating the differentiation of T helper cells.
  • VDR Measu.ri.ng transcriptional, activity of vitamin D receptor.
  • VDR is a member of the steroid receptor superfamily. In its ligand bound state, VDR forms heterodimers with RXR and regulates gene expression by binding to specific hormone response elements.
  • the VDR-RXR heterodimer has been shown to bind to VD-responsive elements (VDRE) of osteocalcin and osteopontin genes to stimulate transcription of these genes.
  • VDRE VD-responsive elements
  • YY1 Measuring transcriptional activity of YY1.
  • YY1 is a ubiquitously
  • YY1 may direct histone deacetylases and histone acetyltransferases to a promoter in order to activate or repress the promoter, thus implicating histone modification in the function of YY1.
  • the response element is a nuclear response element located in the nucleus of the cell. In another embodiment said response element is located on a plasmid in the reporter cell.
  • the assay comprises the preliminary step of transfection of the reporter cells with an expression vector comprising the DNA sequence coding for the reporter gene under the control of the response element.
  • the reporter cells can be transfected with an expression vector comprising the DNA sequence coding for the signal transducing cell surface receptor.
  • the reporter cells can be transfected with an expression vector comprising all elements of the signaling cascade or with different vectors individually expressing the different components.
  • the reporter cells comprise the DNA sequence coding for the reporter gene under the control of the response element, and the DNA sequence coding for signal transducing cell surface receptor.
  • the reporter gene is selected from a gene coding for a fluorescent protein or a gene coding for an enzyme whose catalytic activity can be detected. In one embodiment, the reporter gene is coding for a fluorescent or a luminescent protein. In one embodiment, the reporter gene is coding for green fluorescent protein (GFP) or luciferase. In further embodiments the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), Blue fluorescent protein (BFP, Heim et al. 1994, 1996), a cyan fluorescent variant known as CFP (Heim et al. 1996; Tsien 1998); a yellow fluorescent variant known as YFP (Ormo et al.
  • EGFP enhanced green fluorescent protein
  • the enzyme whose catalytic activity can be detected is selected from the group consisting of luciferase, beta Galactosidase, Alkaline Phosphatase.
  • the reporter gene is encoding for GFP.
  • the reporter gene is encoding for luciferase.
  • the activity of luciferase can be detected by commercially available assays, e.g. by Luciferase 1000 Assay System (or ONE-GloTM Luciferase Assay System (both Promega).
  • the Luciferase 1000 Assay System contains coenzyme A (CoA) besides luciferin as a substrate, resulting in a strong light intensity lasting for at least one minute.
  • CoA coenzyme A
  • a cell lysis buffer was provided separately to the Luciferase 1000 assay system.
  • the ONE-GloTM Luciferase Assay System combines the Luciferase substrate with a cell lysis reagent and also shows a more stable signal.
  • the light which is produced as a by-product of the reaction is collected by the luminometer from the entire visible spectrum.
  • the signal was proportional to the amount of produced luciferase and therefore proportional to the strength of the activation of the NFKB promotor.
  • a Luciferase assay is used wherein the luciferase is secreted from the cells. Hence the assay can be performed witout lysis of the cells.
  • the signal transducing cell surface receptor is functionally linked to a response element.
  • the response element controls the expression of the reporter gene.
  • the signal transducing cell surface receptor and the response element are part of the NF- ⁇ pathway.
  • the signal transducing cell surface receptor is selected from Toll-like receptors, TNF receptors, T cell receptors and B cell receptors; as well as recombinant forms and fragments thereof.
  • Non-limiting examples of antibodies that upon binding to its target result in modulation of the activity of NF- ⁇ are anti-CD3 antibodies, anti-CD40 antibodies, anti-DR5 antibodies, anti-DR4 antibodies, anti-41BB antibodies, anti-Ox40 antibodies and anti-GITR antibodies.
  • the response element is a NF- ⁇ response element.
  • said response element comprises one or more of the following DNA repeats GGGAATTTCC (SEQ ID NO:68), GGGGACTT TCC (SEQ ID NO:69), GGGACTTTCC (SEQ ID NO:70), GGGACTTCC (SEQ ID NO:71), ATTGTAGCGTA (SEQ ID NO:72).
  • said response element comprises 3 to 6, 3 or 6 of the DNA repeats mentioned above.
  • said response element comprises 3 to 6, 3 or 6 of the DNA repeats mentioned above and 1, 2, 3 or 4 additional nucleotides.
  • the reporter cells comprise at least one DNA repeat with a DNA sequence of SEQ ID NO: 68, 69, 70, 71 or 72, wherein the DNA repeat is operatively linked to the reporter gene and wherein the reporter gene is expressed upon binding of the second antigen binding moiety to the signal transducing cell surface receptor.
  • steps iii) and iv) are performed consecutively or simultaneously.
  • the expression of the reporter gene can be directly correlated with the functionality of the antibody to be tested.
  • the amount of light detected from the cells correlates directly with the target antigen binding of the antibody to be tested.
  • the antibody is tested in different concentrations and the half maximal effective concentration (EC50) of reporter gene activation or inhibition is determined.
  • EC50 refers to the concentration of the antibody or ligand at which the antibody or ligand activates or inhibits the reporter gene halfway between the baseline and maximum after a specified exposure time.
  • the EC50 of the dose response curve therefore represents the concentration of the antibody where 50% of its maximal activating or inhibitory effect on the target antigen is observed.
  • the novel assay as described herein is robust, suitable for use in high-throughput format and efficient in terms of hands-on time needed to accomplish the assay. Furthermore, the assay of the present invention tolerates the presence of dead cells in the sample to be analyzed. This is in contrast to cell assays wherein the binding and functionality of an antibody is determined by measuring cell viability or cell death, e.g. a killing assay.
  • the sample to be assayed contains dead cells.
  • the sample to be assayed is a tumor sample, in particular a biopsy of a tumor.
  • the tumor sample contains dead cells, in particular more than 10% of dead cells.
  • the tumor sample contains more than 20%, more than 30%, more than 40% or more than 50% dead cells. Methods to determine the number of dead cells in a cell culture or tissue are well known in the art, as e.g. propidium iodide staining.
  • the antibodies to be tested and the reporter cells can be added to the tumor samples in either order or at the same time.
  • the antibody is diluted in cell culture medium and the tumor sample is added to the cell culture medium containing the diluted antibody in a suitable cell culture format, e.g., in a well of a 24 well plate or in a well of a 96 well plate.
  • the testing medium is a medium that provides conditions for cells to be viable for up to 48 hours. Suitable media are for example Jurkat medium, as outlined in the examples.
  • the assay is performed in a micro titerplate.
  • the microtiterplate is suitable for high throughput screening.
  • the assay of the present invention can be performed in any format that allows for rapid preparation, processing, and analysis of multiple reactions. This can be, for example, in multi-well assay plates (e.g., 24 wells, 96 wells or 386 wells). Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting fluorescent and/or luminescent signals. In one embodiment about 100000 to about 1000000 reporter cells per well of a 24- well plate are provided in step ii).
  • step ii In a preferred embodiment about 300000 to about 700000 cells or about 400000 to about 600000 reporter cells per well of a 24-well plate are provided. In one embodiment about 500000 reporter cells per well of a 24-well plate are provided in step ii). In one embodiment about 10000 to about 100000 cells per well of a 96-well plate are provided in step ii). In a preferred embodiment about 30000 to about 70000 cells or about 40000 to about 60000 cells per well of a 96-well plate are provided. In one embodiment about 50000 reporter cells per well of a 96-well plate are provided in step ii). In one embodiment about 200000 to about 2000000 cells per ml of cell culture medium are provided in step ii). In a preferred embodiment about 600000 to about 1400000 cells or about 800000 to about 1200000 cells per ml of cell culture medium are provided. In one embodiment about 1000000 cells per ml of cell culture medium are provided in step ii).
  • the antibody is provided in step iii) to achieve a final concentration of about 0.001 g/ml to 10 ⁇ g/ml. In further embodiments the antibody is provided in step iii) to achieve a final concentration of about 0.05 g/ml to about 2 g/ml or about 0.1 ⁇ g/ml to about 1 ⁇ g/ml. In further embodiments the antibody is provided in step iii) to achieve a final concentration of about 0.5 ⁇ g/ml. In one embodiment the antibody is provided in step iii) to achieve a final concentration of about 1 nM to about 1000 nM.
  • the antibody is provided in step iii) to achieve a final concentration of about 5 nM to about 200 nM or about 10 nM to about 100 nM. In further embodiments the antibody is provided in step iii) to achieve a final concentration of about 50 nM.
  • the antibody can be diluted in cell culture medium, e.g. in Jurkat medium as described in the example section. The antibody diluted to the final concentration as described herein is added to the tumor sample before or after adding the reporter cells. In one embodiment, the antibody diluted to the final concentration as described herein is added to the tumor sample before adding the reporter cells.
  • the tumor samples are provided in cell culture inserts. In one embodiment, the tumor samples are embedded in Matrigel.
  • the bispecific molecule of the invention binds to CD3. In one specific embodiment the bispecific antibody comprises
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to CD3.
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19.
  • CDR heavy chain complementarity determining region
  • the target antigen is a cell surface receptor. In one embodiment, the target antigen is selected from the group consisting of CEA, Her2, TYRP, EGFR, MCSP, STEAP1, WT1 and FolRl. In one embodiment, the target antigen is FolRl. In one embodiment the bispecific antibody comprises
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to FolRl comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19;
  • CDR heavy chain complementarity determining region
  • a second antigen binding moiety which is a Fab molecule capable of specific binding to CD3, and which comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19.
  • CDR heavy chain complementarity determining region
  • the bispecific antibody comprises (a) a second antigen binding moiety which is a Fab molecule capable of specific binding to FolRl comprising heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
  • a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28.
  • the bispecific antibody comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the first or the second subunit of the Fc domain.
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
  • the bispecific antibody essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain.
  • the Fab light chain of the second antigen binding moiety and the Fab light chain of the first antigen binding moiety may additionally be fused to each other, optionally via a peptide linker.
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • the bispecific antibody essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.
  • the bispecific antibody essentially consists of a first and a second antigen binding moiety, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain.
  • the Fab light chain of the second antigen binding moiety and the Fab light chain of the first antigen binding moiety may additionally be fused to each other.
  • the first and the second antigen binding moieties are conventional Fab molecules comprising a common light chain.
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, optionally via a peptide linker.
  • the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker.
  • the antigen binding moieties may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids.
  • Peptide linkers are known in the art and are described herein.
  • Suitable, non- immunogenic peptide linkers include, for example, (G 4 S) n , (SG 4 ) n , (G 4 S) n or G 4 (SG 4 ) n peptide linkers, "n" is generally a number between 1 and 10, typically between 2 and 4.
  • a particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding moiety to each other is (G 4 S) 2 .
  • linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
  • a bispecific antibody with a single antigen binding moiety capable of specific binding to a target cell antigen is useful, particularly in cases where internalization of the target cell antigen is to be expected following binding of a high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.
  • the bispecific antibody used according to the invention further comprises a third antigen binding moiety capable of specific binding to a target cell antigen.
  • the third antigen binding moiety is a conventional Fab molecule, or a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
  • the third antigen binding moiety is capable of specific binding to the same target cell antigen as the first antigen binding moiety.
  • the second antigen binding moiety is capable of specific binding to CD3, and the first and third antigen binding moieties are capable of specific binding to a target cell antigen.
  • the first and the third antigen binding moiety are identical (i.e. they comprise the same amino acid sequences).
  • the second antigen binding moiety is capable of specific binding to CD3, and the first and third antigen binding moieties are capable of specific binding to FolRl, wherein the first and third antigen binding moieties comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
  • CDR heavy chain complementarity determining region
  • the second antigen binding moiety is capable of specific binding to CD3, and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the first and third antigen binding moieties are capable of specific binding to FolRl, wherein the first and third antigen binding moieties comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
  • CDR heavy chain complementarity determining region
  • the second antigen binding moiety which is a Fab molecule capable of specific binding to CD3, comprising the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 11, the heavy chain CDR 2 of SEQ ID NO: 12, the heavy chain CDR 3 of SEQ ID NO: 13, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged; and the first and third antigen binding moiety each of which is a Fab molecule capable of specific binding to FolRl comprising the heavy chain CDR 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR 2 of S
  • the second antigen binding moiety is capable of specific binding to CD3, and comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28, and the first and third antigen binding moieties are capable of specific binding to FolRl, wherein the second and third antigen binding moieties comprise a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28.
  • the Fc domain of the bispecific antibodies used according to the invention consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule.
  • the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • the two subunits of the Fc domain are capable of stable association with each other.
  • the bispecific antibodies used according to the invention comprises not more than one Fc domain.
  • the Fc domain of the bispecific antibody is an IgG Fc domain.
  • the Fc domain is an IgGl or IgG4, Fc domainln a particular embodiment the Fc domain is an IgGl Fc domain.
  • the Fc domain is an IgG4 Fc domain.
  • the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG 4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
  • the Fc domain is a human Fc domain.
  • Bispecific antibodies used according to the invention comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity bispecific antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bispecific antibodies a modification promoting the association of the desired polypeptides.
  • the Fc domain of the bispecific antibodies used according to the invention comprise a modification promoting the association of the first and the second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • said modification is a so-called "knob-into-hole” modification, comprising a "knob” modification in one of the two subunits of the Fc domain and a "hole” modification in the other one of the two subunits of the Fc domain.
  • the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W)
  • T366W tryptophan residue
  • Y407V valine residue
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C)
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C).
  • the antigen binding moiety capable of binding to CD3 is fused (optionally via the antigen binding moiety capable of binding to the target cell antigen) to the first subunit of the Fc domain (comprising the "knob" modification).
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • the bispecific antibody of step iii) additionally comprises c) a masking moiety covalently attached to the second antigen binding moiety through a protease-cleavable linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety.
  • a masking moiety covalently attached to the second antigen binding moiety through a protease-cleavable linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety.
  • protease is expressed by the tumor sample.
  • the protease capable of cleaving the protease-cleavable linker is selected from the group consisting of metalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine protease, e.g., urokinase-type plasminogen activator and Matriptase, cysteine protease, aspartic protease, and cathepsin protease.
  • the protease is Matriptase. Expression of protease is known in the art to be indicative of malignant tumors. Accordingly, in one embodiment, protease expression is indicative for a malginant tumor.
  • the reporter gene is expressed by the reporter cells upon binding of the first antigen binding moiety to the target antigen on the tumor cells in the tumor sample and cleavage of the protease-cleavable linker by a protease expressed by the tumor cells and subsequent binding of the second antigen binding moiety to the signal transducing cell surface receptor.
  • the second antigen binding moiety Upon cleavage of the linker, the second antigen binding moiety is revealed and binds to the signal transducing cell surface receptor, wherein expression of the reporter gene initiated. Accordingly, expression of the reporter gene is indicative for target antigen and protease expression in the tumor, wherein protease expression is indicative for a malignant tumor.
  • the anti-idiotype masking moiety binds to the idiotype of the second antigen binding moiety.
  • the anti-idiotype masking moiety has a KD of about 1 nM to about 8 nM, in particular as determined by Surface Plasmon Resonance (SPR).
  • anti-idiotype mask has a KD of about 2 nM at 37 °C as determined by SPR.
  • the masking moiety recognizes the idiotype of the second antigen binding moiety capable of specific binding to a CD3, e.g., a human CD3.
  • the masking moiety recognizes the idiotype of the second antigen binding moiety capable of binding to a target cell antigen.
  • the second antigen binding moiety is capable of specific binding to CD3.
  • the second antigen binding moiety capable of specific binding to CD3 comprises an idiotype.
  • the masking moiety of the protease-activatable T cell activating bispecific molecule is covalently attached to the second antigen binding moiety.
  • the masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety.
  • the masking moiety is covalently attached to the light chain variable region of the second antigen binding moiety. This covalent bond is separate from the specific binding, which is preferably non-covalent, of the masking moiety to the idiotype first antigen binding site.
  • the idiotype of the second antigen binding moiety comprises its variable region.
  • the masking moiety binds to amino acid residues that make contact with CD3 when the second antigen biding moiety is bound to CD3.
  • the masking moiety is not the cognate antigen or fragments thereof of the second antigen binding moiety, i.e., the masking moiety is not a CD3 or fragments thereof.
  • the masking moiety is an anti-idiotypic antibody or fragment thereof.
  • the masking moiety is an anti-idiotypic scFv. Exemplary embodiments of masking moieties which are anti-idiotypic scFv, and protease activatable T cell activating molecules comprising such masking moieties, are described in detail in the examples.
  • the masking moiety masks a CD3-binding moiety and comprises at least one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
  • the masking moiety comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
  • the idiotype-specific polypeptide is an anti-idiotype scFv. In one embodiment the idiotype-specific polypeptide is covalently attached to the molecule through a linker. In one embodiment the idiotype-specific polypeptide is covalently attached to the molecule through more than one linker.
  • the idiotype- specific polypeptide is covalently attached to the molecule through two linkers.
  • the linker is a peptide linker.
  • the linker is a protease- cleavable linker.
  • the protease-cleavable linker comprises the sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44.
  • the protease-cleavable linker comprises at least one protease recognition site.
  • the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59.
  • the protease cleavable linker comprises a protease recognition sequence.
  • the protease recognition sequence is selected from the group consisting of:
  • VHMPLGFLGPGRSRGSFP SEQ ID NO:46
  • VHMPLGFLGPRQARVVNG SEQ ID NO:50
  • QARAK SEQ ID NO:56
  • VHMPLGFLGPPMAKK (SEQ ID NO:57);
  • the protease is selected from the group consisting of metalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine protease, e.g., urokinase-type plasminogen activator and Matriptase, cysteine protease, aspartic protease, and cathepsin protease.
  • MMP matrix metalloproteinase
  • ADAM Disintegrin And Metalloproteinase
  • the protease is selected from the group consisting of metalloproteinase, serine protease, cysteine protease, aspartic proteases, and cathepsin protease.
  • the protease is a metalloproteinase.
  • the metalloproteinase is a matrix metalloproteinase (MMP), in particular MMP9 or MMP2.
  • MMP matrix metalloproteinase
  • the protease is a serine protease.
  • the serine protease is Matriptase.
  • a protease cleaves the protease-cleavable linker, wherein the second antigen binding moiety is unconcealed.
  • the protease as described herein is expressed by the tumor cells.
  • the protease is expressed in the tumor sample, in particular in the biopsy as described herein.
  • the protease is expressed in a tumor tissue sample, in particular a biopsy from a patient.
  • the expression of the reporter gene is indicative for protease expression in the tumor sample.
  • antibodies comprising a masking moiety connected to the second antigen binding moiety through a protease-cleavable linker are used to detect protease expression in tumor samples.
  • the target antigen of the protease-activatable bispecific antibody is a cell surface receptor.
  • the target antigen is selected from the group consisting of CEA, Her2, TYRP, EGFR, MCSP, STEAP1, WT1 and FolRl.
  • the target antigen is FolRl.
  • the protease-activatable bispecific antibody used according to the invention comprises at least one antigen binding moiety that is specific for FolRl further comprises an anti-idiotypic CD3 scFv comprising at least one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
  • an anti-idiotypic CD3 scFv comprising at least one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
  • the anti- idiotypic scFv comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
  • the protease-activatable bispecific antibody used according to the invention comprises at least one antigen binding moiety that is specific for FolRl further comprises an anti-idiotypic CD3 scFv comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29.
  • the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 29.
  • the assay of the present invention is able to assess both binding to a target antigen as described herein and expression of a protease as described herein.
  • the binding to the target antigen and the expression of the protease are determined in the same vial.
  • the assay of the present invention is used for selection of protease-cleavable linkers suitable for the treatment of a tumor.
  • the protease-activatable bispecific antibody used according to the invention comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6 and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7.
  • the protease-activatable bispecific antibody used according to the invention comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 6 and the polypeptide sequence of SEQ ID NO: 7.
  • the protease-activatable bispecific antibody used according to the invention comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7 and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.
  • the protease-activatable bispecific antibody used according to the invention comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 7 and the polypeptide sequence of SEQ ID NO: 10.
  • the protease-activatable bispecific antibody used according to the invention comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7 and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8.
  • the protease-activatable bispecific antibody used according to the invention comprises the polypeptide sequence of SEQ ID NO: 5, the polypeptide sequence of SEQ ID NO: 7 and the polypeptide sequence of SEQ ID NO: 8.
  • the protease-activatable bispecific antibody used according to the invention comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.
  • the protease-activatable bispecific antibody used according to the invention comprises the polypeptide sequence of SEQ ID NO: 3, the polypeptide sequence of SEQ ID NO: 4 and the polypeptide sequence of SEQ ID NO: 5.
  • a method for selecting a bispecific antibody for the treatment of a tumor wherein the bispecific antibody comprises: a. a first antigen binding moiety capable of specific binding to a target antigen; and
  • a second antigen binding moiety capable of specific binding to a signal transducing cell surface receptor
  • the method comprises determining the presence of a target antigen in a tumor sample according to the method as described herein and wherein the bispecific antibody is selected for treatment of the tumor if expression of the reporter gene is detected.
  • the bispecific antibody additionally comprises c) a masking moiety covalently attached to the second antigen binding moiety through a pro tease-cleav able linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety as described herein.
  • a masking moiety covalently attached to the second antigen binding moiety through a pro tease-cleav able linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety as described herein.
  • Bispecific antibodies targeting different antigens on the same or even different cells are considered to overcome some of the current challenges in cancer therapy.
  • Some of these construct bind to antigens on different cells, e.g., to a target antigen on a cancer cell and an immuno stimulatory antigen on an effector cell, in particular on T cells.
  • the target antigen is supposed to direct the bispecific antibody to the tumor tissue whereas the immuno stimulatory antigen activates the effector cells which leads to efficient destruction of the tumor cells by the effector cells, e.g., T cells.
  • Some constructs comprise activatable immunomodulatory moieties which are unconcealed upon binding to the tumor. This can be done as described herein by concealing the immunomodulatory moiety with a masking moiety attached to the immunomodulatory moiety through a protease-cleavable linker. These constructs are described herein to as being protease- activatable.
  • the tumor cells For efficient lysis of a tumor, the tumor cells must express the target antigen in suitable amount for the antibody to bind efficiently to the tumor cells. Additionally, for protease- activatable constructs with concealed moieties, the tumor cells must also express a tumor tissue specific protease in suitable amounts to efficiently cleave the protease-cleavable linker between the masking moity and the masked moiety.
  • the method according to the present invention provides an assay to assess if a bispecific antibody is suitable for the treatment of a tumor by assessing a tumor sample, e.g., a tumor biopsy for target antigen binding and/or protease expression.
  • the expression of the reporter gene measured in a method according to the invention is indicative for a suitable bispecific antibody for the treatment of a tumor, wherein the tumor sample is a tumor biopsy from a patient and wherein the bispecific antibody is a candidate antibody for treatment of the tumor.
  • the assay of the present invention is used for selection of protease-cleavable linkers suitable for the treatment of a tumor. The assessment can be done in hight-throughput format as described herein, i.e. a multitude of candidate antibodies for treatment of a tumor can be assessed in parallel. The assay is robust and tolerates the presence of dead cells in the tumor sample. Bispecific antibodies selected for the treatment of a tumor can be used in therapeutic methods as described herein.
  • Protease- cleavable linkers suitable for the treatment of a tumor as selected using the assay of the invention can be included in novel or known bispecific antibodies for the treatment of cancer. Any of bispecific antibodies selected for the treatment of a tumor as described herein, may be used in therapeutic methods. Bispecific antibodies selected for the treatment of a tumor as described herein can be used as immunotherapeutic agents, for example in the treatment of cancers. For use in therapeutic methods, bispecific antibodies selected according to the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • bispecific antibodies selected for the treatment of a tumor according to the method of the invention are provided.
  • the selected bispecific antibodies for use as a medicament are provided.
  • selected bispecific antibodies of the invention for use in treating a disease are provided.
  • selected bispecific antibodies of the invention for use in a method of treatment are provided.
  • the invention provides a bispecific antibody selected as described herein for use in the treatment of a disease in an individual in need thereof.
  • the invention provides a bispecific antibody for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the selected bispecific antibody.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
  • the invention provides a bispecific antibody selected as described herein for use in inducing lysis of a target cell, particularly a tumor cell.
  • the invention provides a bispecific antibody for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the selected bispecific antibody to induce lysis of a target cell.
  • An "individual" according to any of the above embodiments is a mammal, preferably a human.
  • the invention provides for the use of a bispecific antibody selected for the treatment of a tumor as described herein in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of a disease in an individual in need thereof.
  • the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti- cancer agent if the disease to be treated is cancer.
  • the medicament is for inducing lysis of a target cell, particularly a tumor cell.
  • the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the medicament to induce lysis of a target cell.
  • An "individual" according to any of the above embodiments may be a mammal, preferably a human.
  • a tumor sample, e.g., a tumor biopsy, of the individual is assessed using the methods according to the invention to find a suitable bispecific antibody for the treatment of the tumor.
  • the invention provides a method for treating a disease.
  • the method comprises selecting a bispecific antibody for the treatment of the disease according to the methods as described herein and administering to an individual having such disease a therapeutically effective amount of the selected bispecific antibody.
  • a composition is administered to said invididual, comprising the selected bispecific antibody in a pharmaceutically acceptable form.
  • the disease to be treated is a proliferative disorder.
  • the disease is cancer.
  • the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
  • An "individual" according to any of the above embodiments may be a mammal, preferably a human.
  • the invention provides a method for inducing lysis of a target cell, particularly a tumor cell.
  • the method comprises contacting a target cell with a bispecific antibody selected according to the invention in the presence of a T cell, particularly a cytotoxic T cell.
  • a method for inducing lysis of a target cell, particularly a tumor cell, in an individual is provided.
  • the method comprises administering to the individual an effective amount of a bispecific antibody to induce lysis of a target cell.
  • an "individual" is a human.
  • the disease to be treated is a proliferative disorder, particularly cancer.
  • Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.
  • the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • bispecific antibody may not provide a cure but may only provide partial benefit.
  • a physiological change having some benefit is also considered therapeutically beneficial.
  • an amount of bispecific antibody that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount".
  • the subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.
  • an effective amount of a selected bispecific antibody according to the invention is administered to a cell.
  • a therapeutically effective amount of a bispecific antibody is administered to an individual for the treatment of disease.
  • the appropriate dosage of a bispecific antibody (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of T cell activating bispecific antibody, the severity and course of the disease, whether the T cell activating bispecific antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the bispecific antibody, and the discretion of the attending physician.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • the selected bispecific antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g., 0.1 mg/kg - 10 mg/kg) of bispecific antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the T cell activating bispecific antibody would be in the range from about 0.005 mg/kg to about 10 mg/kg.
  • a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligram/kg body weight, about 10 milligram/kg body weight, about 50 milligram/kg body weight, about 100 milligram/kg body weight, about 200 milligram/kg body weight, about 350 milligram/kg body weight, about 500 milligram/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 microgram/kg body weight to about 500 milligram/kg body weight, etc. can be administered, based on the numbers described above.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the bispecific antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • the selected bispecific antibody according to the invention will generally be used in an amount effective to achieve the intended purpose.
  • the selected bispecific antibodys, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. Dosage amount and interval may be adjusted individually to provide plasma levels of the bispecific antibodys which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
  • a therapeutically effective dose of the selected bispecific antibodys described herein will generally provide therapeutic benefit without causing substantial toxicity.
  • Toxicity and therapeutic efficacy of a bispecific antibody can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose lethal to 50% of a population) and the ED 50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 /ED 50 .
  • Bispecific antibodies that exhibit large therapeutic indices are preferred.
  • the selected bispecific antibody according to the present invention exhibits a high therapeutic index.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans.
  • the dosage lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
  • the attending physician for patients treated with selected bispecific antibodys according to the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • An in vitro method for determining the presence of a target antigen in a tumor sample comprising the steps of:
  • reporter cells comprising a reporter gene under the control of a
  • a bispecific antibody comprising:
  • bispecific antibody additionally comprises:
  • a masking moiety covalently attached to the second antigen binding moiety through a protease-cleavable linker, wherein the masking moiety is capable of specific binding to the idiotype of the second antigen binding moiety thereby reversibly concealing the second antigen binding moiety;
  • sample is a tumor tissue sample, in particular a biopsy from a patient.
  • the reporter gene is coding for green fluorescent protein (GFP) or luciferase. 0.
  • the reporter cells comprise the DNA sequence coding for the reporter gene under the control of the response element, and the DNA sequence coding for the signal transducing cell surface receptor.
  • the reporter cells comprise at least one DNA repeat with a DNA sequence of SEQ ID NO: 68, 69, 70, 71 or 72, wherein the DNA repeat is operatively linked to the reporter gene and wherein the reporter gene is expressed upon binding of the second antigen binding moiety to the signal transducing cell surface receptor.
  • the second antigen binding moiety is capable of specific binding to CD3s.
  • the protease- cleavable linker comprises a protease recognition sequence.
  • the protease recognition sequence is selected from the group consisting of:
  • VHMPLGFLGPGRSRGSFP SEQ ID NO:46
  • VHMPLGFLGPRQARVVNG (SEQ ID NO:50);
  • VHMPLGFLGPPMAKK (SEQ ID NO:57);
  • X is any amino acid.
  • the protease is selected from the group consisting of metalloproteinase, serine protease, cysteine protease, aspartic proteases, and cathepsin protease.
  • the metalloproteinase is a matrix metalloproteinase (MMP), particularly MMP9 or MMP2.
  • MMP matrix metalloproteinase
  • Matriptase The method according to any one of embodiments 4 to 27, wherein the masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety.
  • the first and the second antigen binding moieties are conventional Fab molecules comprising a common light chain.
  • the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, optionally via a peptide linker.
  • 34 The method according to any one of embodiments 32 or 33, wherein the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker.
  • the bispecific antibody comprises a third antigen binding moiety capable of specific binding a tumor antigen.
  • Fab moiety is a conventional Fab molecule, or a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
  • bispecific antibody additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • Fc domain is an IgG, specifically an IgGl or IgG4, Fc domain.
  • domain is a human Fc domain.
  • the target antigen is a cell surface receptor.
  • the target antigen is FolRl.
  • the target antigen is a peptide bound to a molecule of the human major histocompatibility complex (MHC).
  • MHC human major histocompatibility complex
  • a second antigen binding moiety capable of specific binding to a signal transducing cell surface receptor
  • the method comprises determining the presence of a target antigen in a tumor sample according to the method of any one of embodiments 1 to 45 and wherein the bispecific antibody is selected for treatment of the tumor if expression of the reporter gene is detected.
  • ID 8409 "FolRl 2+1 IgG, classic format (anti idiotypic scFv 4.32.63 - non cleavable linker - CD3 - N-terminal fused to FolRl VH - inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and non cleavable GS linker.”
  • FIG. 2C shows that
  • variable domains were subcloned in frame with the pre-inserted domains into the respective recipient mammalian expression vector.
  • Protein expression is driven by an MPSV promoter and a synthetic polyA signal sequence is present at the 3' end of the CDS.
  • each vector contains an EBV OriP sequence.
  • the molecules were produced by co-transfecting HEK293-EBNA cells growing in suspension with the mammalian expression vectors using polyethylenimine (PEI).
  • PEI polyethylenimine
  • HEK293 EBNA cells were cultivated in serum free ExCell culture medium containing 6 mM L-glutamine and 250 mg / 1 G418.
  • 600 ml tubespin flasks maximum. working volume 400 ml
  • 800 million HEK293 EBNA cells were seeded 24 hours before transfection without G418.
  • transfection 800 mio cells were centrifuged for 5 min at 210 x g and supernatant was replaced by 40 ml pre- warmed CD CHO medium containing 6mM L-Glutamine.
  • Expression vectors were mixed with 40 ml CD CHO medium containing 6mM L-Glutamine to a total amount of 400 ⁇ g DNA. After addition of 1080 ⁇ PEI solution (2.7 ⁇ g/ml) the mixture was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C in an incubator with a 5% C0 2 atmosphere.
  • Target protein was concentrated with Amicon®Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a volume of 4 ml maximum prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM NaCl, 0.01% Tween pH 6.0.
  • the protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm divided by the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the molecules after the final purification step were analyzed by CE-SDS analyses in the presence and absence of a reducing agent.
  • the Caliper LabChip GXII system (Caliper Lifescience) was used according to the manufacturer's instruction.
  • the aggregate content of the molecules was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HP04, 125 mM NaCl, 200 mM L- arginine monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25 °C.
  • Capillary Electrophoresis SDS analysis of different TCB molecules Purity and molecular weight of the molecules after the final purification step were analyzed by CE-SDS analyses in the presence and absence of a reducing agent.
  • the Caliper LabChip GXII system (Caliper Lifescience) was used according to the manufacturer's instruction. Comparison of untreated molecules (stored at 4 °C), treated molecules (treated with appropriate recombinant protease (R&D Systems) for 24 h at 37 °C and molecule incubated for 72 h at 37 °C.
  • Jurkat NFAT activation assay for comparison of different formats and linkers of protease-activated TCB.
  • Jurkat- NFAT reporter cell line (Promega) is a human acute lymphatic leukemia reporter cell line with a NFAT promoter, expressing human CD3s. If the TCB binds the tumor target and the CD3 binder (crosslinkage) binds the CD3E Luciferase expression can be measured in Luminescence after addition of One- Glo substrate (Promega).
  • 20.000 target cells were seeded in 96-well white walled clear bottom plate (Greiner BioOne) in 50 ul / well Jurkat medium (RPMI1640, 2g/l Glucose, 2 g/1 NaHCCe, 10 % FCS, 25 mM HEPES, 2 mM L-Glutamin, 1 x NEAA, 1 x Sodium- pyruvate) without Hygromycine. Plates were incubated for about 20 hours at 37 °C. Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell. Cells were resuspended in Jurkat medium without Hygromycine and 50 ⁇ per well (50.000 cells / well) were added.
  • Jurkat medium RPMI1640, 2g/l Glucose, 2 g/1 NaHCCe, 10 % FCS, 25 mM HEPES, 2 mM L-Glutamin, 1 x NEAA, 1 x Sodium- pyruvate
  • the E:T ratio was 2.5: 1 (based on cell number seeded).
  • Antibodies were diluted in Jurkat medium without Hygromycine and 50 ul / well were added. Cells were incubated at 37 °C for 6 h in a humidified incubator before they were taken out of the incubator for about 10 min to adapt to room temperature prior to Luminescence read out. 50 ⁇ /well of ONE-Glo solution were added to wells and incubated for 10 min at room temperature in the dark. Luminescence was detected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/well as detection time.
  • the protease-activated TCBs were treated with rhMatriptase/ST14 (R&D Systems) for about 20 h at 37 °C.
  • Comparison of the pretreated protease-activated TCB (8364, grey filled squares) and FolRl TCB (black triangles pointing down) showed that potency after cleavage is recovered completely.
  • No Luminescence was detectable for cells incubated with the masked TCB (containing a GS non cleavable linker, grey triangles pointing up) and the non-targeted TCB control (empty triangle pointing down) for both cell lines in this concentration range.
  • the dotted line shows the Luminescence of target cells and effector cells without any TCB (FIGURES 4A-B).
  • the intention of this assay was to show tumor target antigen (FolRl) expression and activity of tumor specific proteases like MMP9, Matriptase or Cathepsin in human tumor samples.
  • TulRl tumor target antigen
  • Jurkat-NFAT reporter cell line is a human acute lymphatic leukemia reporter cell line with a NFAT promoter, expressing human CD3s. Luciferase expression can be measured, if the T cell bispecific molecule binds the tumor target and the CD3s (crosslinkage). Luminescence is measured after addition of One-Glo substrate (Promega). Primary tumor samples were received from Indivumed GmbH, Germany. Samples were shipped over night in transport medium. About 24 h after surgery the sample was cut in small pieces. First method (FIGURE 5):
  • 24- well plate was prepared by inserting one Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 ⁇ (PICM01250, MerckMillipore) in each well.
  • Antibodies were diluted in Jurkat medium without Hygromycine but with 1.5 X penicillin/strep tomycine solution. 400ul were added inside the well and 600ul were added outside the filter. Two to three pieces of human tumor were added to each well and incubated for 48 hours at 37 °C, 5 % C02.
  • Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell.
  • Cells were centrifuged at 350 x g, 7 min before they were resuspended in Jurkat medium without Hygromycine and 500 ⁇ per well (500.000 cells / well) were added. Plate was incubated for 5 h at 37 °C in a humidified incubator before it was taken out for Luminescence read out. 500 ⁇ of ONE-Glo solution was added to each well and incubated for 10 min at room temperature in the dark. Luminescence was detected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/well as detection time.
  • 96-well white walled, flat (clear) bottom plate was prepared by adding 18ul cold Matrigel (Matrigel (734-1101, Corning/VWR). Plate was incubated for 2 min at 37 °C before tumor pieces were added (triplicates). 33 ul of cold Matrigel were added per well and plate was incubated again for 2 min at 37 °C. 50 ul of antibody dilution (in Jurkat medium without Hygromycine but containing 2X Penicillin/Streptomycine) was added per well and plate was incubated for about 48 hours at 37 °C, 5 % C02.
  • Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell.
  • Cells were centrifuged at 350 x g, 7 min before they were resuspended in Jurkat medium without Hygromycine and 50 ⁇ per well (50.000 cells / well) were added. Plate was incubated for 5 h at 37 °C in a humidified incubator before it was taken out for Luminescence read out. 80 ul of each well were transferred into a white walled 96-well plate. 27 ⁇ /well of ONE-Glo solution were added to each well and incubated for 10 min at room temperature in the dark. Luminescence was detected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/well as detection time.
  • Jurkat NFAT reporter cells are activated after incubation with FolRl TCB (6298) and tumor samples.
  • Protease-activated FolRl TCBs (8363, 8408) and control TCBs (8409, 7235) do not induce Luciferase expression.
  • the dotted line indicates the baseline Luminescence for Jurkat NFAT cells co-incubated with tumor (FIGURE 5&6).
  • Luminescence can be detected for the Jurkat NFAT cells incubated with the malignant tumor sample ( Figure 6) and the protease-activated FolRl TCB (MMP9- Matriptase, 8364). However no Jurkat NFAT activation can be measured for the benign tumor sample ( Figure 5) and the protease-activated FolRl TCB (MMP9-Matriptase, 8364). Exainple 5
  • Tumors derived from PDXs were excised and cut into the smallest pieces possible, incubated for 1 h with 200 U/ml Collagenase 1 A Sigma), washed, filtered through 100 ⁇ strainers (Corning ) and counted. Single target cells were co-cultured with effector cells at an 5: 1 EffectonTarget (E:T) ratio in 96- wel l V bottom plates, in the presence of p95HER2-TCB. The plate was incubated for 16 h at 37 °C in a humidified incubator before it was taken out for Luminescence read out as described before.
  • E:T EffectonTarget
  • VH_3-23(12) VL7- GIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAK
  • VH3_23-VH12 TFGCGTKLEIKGGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGSEVQ
  • VH CHI Fc hole GGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCA
  • AFGAGTKT ET KRTVAAPSVF1FPPSDRKLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

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Abstract

La présente invention concerne une nouvelle analyse fondée sur des cellules afin de déterminer une expression d'antigène dans des échantillons de tumeur primaire. Le procédé concerne en outre la détermination de l'expression de l'antigène et de la protéase dans des échantillons de tumeur primaire. Le procédé permet une détermination robuste de l'expression de l'antigène et/ou de la protéase sans qu'il soit nécessaire de digérer les échantillons de tumeur. Le procédé permet en outre la sélection d'anticorps et la sélection de lieurs clivables par protéase pour le traitement de tumeurs.
EP17825147.6A 2016-12-13 2017-12-11 Procédé de détermination de la présence d'un antigène cible dans un échantillon de tumeur Pending EP3555620A1 (fr)

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JP7022123B2 (ja) 2016-09-30 2022-02-17 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト Ｃｄ３に対する二重特異性抗体
US20210333286A1 (en) * 2020-04-24 2021-10-28 Glympse Bio, Inc. Activity sensor controls
EP3915576A1 (fr) * 2020-05-28 2021-12-01 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Récepteurs d'antigène chimérique spécifiques pour p95her2 et leurs utilisations
PE20231552A1 (es) * 2020-06-19 2023-10-03 Hoffmann La Roche Anticuerpos biespecificos para linfocitos t activados por proteasa

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